Methods that are most frequently used to obtain a gene sample for gene analysis include a polymerase chain reaction method based on DNA polymerase. This method has an advantage in that it can accurately amplify only the desired region of a desired gene by selectively controlling and designing the lengths and nucleotide sequences of primers capable of binding to the template DNA. However, in this method, only one gene of interest can be amplified by a single reaction, and thus when the number of the genes to be amplified is large, there is a shortcoming in that the same operation should be repeatedly performed.
To analyze a number of gene regions at the same time, a multiplex polymerase chain reaction method is widely used in which several polymerase chain reactions are performed in a single tube. However, in this method, a number of primers are simultaneously used in a single tube, and thus a cross-reaction between the primers occurs. For this reason, there is a shortcoming in that the number of gene regions that can be amplified at the same time is limited. In addition, there are shortcomings in that it requires large amounts of effort and time to find reaction conditions and in that good results in terms of sensitivity and specificity cannot be obtained (Hardenbol et al., Nat. Biotechnol., 21:673, 2003).
In recent years, studies have been actively conducted to enable high-throughput analysis by amplifying a number of gene regions using universal primers without using multiplex polymerase chain reactions. Typical technologies include SNPlex capable of analyzing the single nucleotide polymorphisms (SNPs) of several gene regions at the same time, a Goldengate assay, molecular inversion probes (MIPs) and the like.
SNPlex is a method in which DNA is purified using exonuclease after an oligonucleotide ligation assay (OLA) and amplified by a polymerase chain reaction using universal primer nucleotide sequences located at both ends of the probe, after which the amplification products are analyzed in a DNA chip using a ZipCode nucleotide sequence included in the probe (Tobler et al., J. Biomol. Tech., 16:398, 2005).
The Goldengate assay is a method in which an allele-specific primer extension reaction is performed on a genomic DNA immobilized on a solid surface using an upstream probe, after which the DNA is ligated with a downstream probe and washed to remove probes not ligated to the DNA. Then, the DNA is amplified using universal primer nucleotide sequences included in the probes, like the case of SNPlex, and the PCR amplification products are analyzed in Illumina BeadChip (Shen et al., Mutat. Res., 573:70, 2005).
The molecular inversion probe (MIP) assay is a method in which gap-ligation is performed using a padlock probe, after which probes and genomic DNA, not ligated to the DNA, are removed using exonucelase, and the padlock probe is linearized using uracil-N-glycosylase. Then, the DNA is amplified by a polymerase chain reaction using universal primer nucleotide sequences included in the probe, and the amplification products are hybridized to the GenFlex tag array chip (Affymetrix) to analyze the single nucleotide polymorphisms (Hardenbol et al., Nat. Biotechnol., 21:673, 2003).
However, these methods have problems in that, because portions of reaction products in a first tube are transferred and reacted in a second tube or several kinds of enzymes should be used, contamination between different samples can occur and experimental methods are complex.
Accordingly, the present inventors have made extensive efforts to solve the above-described problems occurring in the prior art, and as a result, have found that, when probes comprising a nucleotide sequence complementary to a gene of interest is ligated with each other by ligase and subjected to an extension reaction to prepare a template probe, which is then subjected to a separation process using a tag and amplified using universal primers, the template probe for the gene of interest can be amplified using the universal primers in a more accurate and rapid manner than conventional methods, thereby completing the present invention.